Biocatalytic Process Optimization Printed Edition of the Special Issue Published in Catalysts www.mdpi.com/journal/catalysts Chia-Hung Kuo and Chwen-Jen Shieh Edited by Biocatalytic Process Optimization Biocatalytic Process Optimization Editors Chia-Hung Kuo Chwen-Jen Shieh MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Chia-Hung Kuo National Kaohsiung University of Science and Technology Taiwan Chwen-Jen Shieh National Chung-Hsing University Taiwan Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Catalysts (ISSN 2073-4344) (available at: https://www.mdpi.com/journal/catalysts/special issues/ biocata process optimiz). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. 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Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Chia-Hung Kuo and Chwen-Jen Shieh Biocatalytic Process Optimization Reprinted from: Catalysts 2020 , 10 , 1303, doi:10.3390/catal10111303 . . . . . . . . . . . . . . . . . 1 Chan-Su Rha, Shin-Woo Kim, Kyoung Hee Byoun, Yong Deog Hong and Dae-Ok Kim Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme Reprinted from: Catalysts 2019 , 9 , 861, doi:10.3390/catal9100861 . . . . . . . . . . . . . . . . . . . 7 Bianca Grabner, Yekaterina Pokhilchuk and Heidrun Gruber-Woelfler DERA in Flow: Synthesis of a Statin Side Chain Precursor in Continuous Flow Employing Deoxyribose-5-Phosphate Aldolase Immobilized in Alginate-Luffa Matrix Reprinted from: Catalysts 2020 , 10 , 137, doi:10.3390/catal10010137 . . . . . . . . . . . . . . . . . 27 I-Chun Cheng, Jin-Xian Liao, Jhih-Ying Ciou, Li-Tung Huang, Yu-Wei Chen and Chih-Yao Hou Characterization of Protein Hydrolysates from Eel ( Anguilla marmorata ) and Their Application in Herbal Eel Extracts Reprinted from: Catalysts 2020 , 10 , 205, doi:10.3390/catal10020205 . . . . . . . . . . . . . . . . . . 43 Chengcheng Jiang, Zhen Liu, Jianan Sun, Changhu Xue and Xiangzhao Mao A Novel Route for Agarooligosaccharide Production with the Neoagarooligosaccharide-Producing β -Agarase as Catalyst Reprinted from: Catalysts 2020 , 10 , 214, doi:10.3390/catal10020214 . . . . . . . . . . . . . . . . . . 55 Yumei Hu, Jian Min, Yingying Qu, Xiao Zhang, Juankun Zhang, Xuejing Yu and Longhai Dai Biocatalytic Synthesis of Calycosin-7-O- β -D-Glucoside with Uridine Diphosphate–Glucose Regeneration System Reprinted from: Catalysts 2020 , 10 , 258, doi:10.3390/catal10020258 . . . . . . . . . . . . . . . . . . 65 Chen-Fu Chung, Shih-Che Lin, Tzong-Yuan Juang and Yung-Chuan Liu Shaking Rate during Production Affects the Activity of Escherichia coli Surface-Displayed Candida antarctica Lipase A Reprinted from: Catalysts 2020 , 10 , 382, doi:10.3390/catal10040382 . . . . . . . . . . . . . . . . . . 77 Li-Hua Du, Rui-Jie Long, Miao Xue, Ping-Feng Chen, Meng-Jie Yang and Xi-Ping Luo Continuous-Flow Synthesis of β -Amino Acid Esters by Lipase-Catalyzed Michael Addition of Aromatic Amines Reprinted from: Catalysts 2020 , 10 , 432, doi:10.3390/catal10040432 . . . . . . . . . . . . . . . . . . 93 Andrey S. Aksenov, Irina V. Tyshkunova, Daria N. Poshina, Anastasia A. Guryanova, Dmitry G. Chukhchin, Igor G. Sinelnikov, Konstantin Y. Terentyev, Yury A. Skorik, Evgeniy V. Novozhilov and Arkady P. Synitsyn Biocatalysis of Industrial Kraft Pulps: Similarities and Differences between Hardwood and Softwood Pulps in Hydrolysis by Enzyme Complex of Penicillium verruculosum Reprinted from: Catalysts 2020 , 10 , 536, doi:10.3390/catal10050536 . . . . . . . . . . . . . . . . . . 107 v Chia-Hung Kuo, Chun-Yung Huang, Chien-Liang Lee, Wen-Cheng Kuo, Shu-Ling Hsieh and Chwen-Jen Shieh Synthesis of DHA/EPA Ethyl Esters via Lipase-Catalyzed Acidolysis Using Novozym R © 435: A Kinetic Study Reprinted from: Catalysts 2020 , 10 , 565, doi:10.3390/catal10050565 . . . . . . . . . . . . . . . . . . 133 Daniel Eggerichs, Carolin M ̈ ugge, Julia Mayweg, Ulf-Peter Apfel and Dirk Tischler Enantioselective Epoxidation by Flavoprotein Monooxygenases Supported by Organic Solvents Reprinted from: Catalysts 2020 , 10 , 568, doi:10.3390/catal10050568 . . . . . . . . . . . . . . . . . . 147 Ilona Sadauskiene, Arunas Liekis, Inga Staneviciene, Rima Naginiene and Leonid Ivanov Effects of Long-Term Supplementation with Aluminum or Selenium on the Activities of Antioxidant Enzymes in Mouse Brain and Liver Reprinted from: Catalysts 2020 , 10 , 585, doi:10.3390/catal10050585 . . . . . . . . . . . . . . . . . . 161 Magdalena Rychlicka, Natalia Niezgoda and Anna Gliszczy ́ nska Development and Optimization of Lipase-Catalyzed Synthesis of Phospholipids Containing 3,4-Dimethoxycinnamic Acid by Response Surface Methodology Reprinted from: Catalysts 2020 , 10 , 588, doi:10.3390/catal10050588 . . . . . . . . . . . . . . . . . . 173 Liwei Zhang, Yuxiao Lu, Xiaobin Feng, Qinghong Liu, Yuanhui Li, Jiamin Hao, Yanqiong Wang, Yongqiang Dong and Huimin David Wang Hepatoprotective Effects of Pleurotus ostreatus Protein Hydrolysates Yielded by Pepsin Hydrolysis Reprinted from: Catalysts 2020 , 10 , 595, doi:10.3390/catal10060595 . . . . . . . . . . . . . . . . . . 187 Andreea Veronica Botezatu (Dediu), Georgiana Horincar, Ioana Otilia Ghinea, Bianca Furdui, Gabriela-Elena Bahrim, Vasilica Barbu, Fanica Balanescu, Lidia Favier and Rodica-Mihaela Dinica Whole-Cells of Yarrowia lipolyti ca Applied in “One Pot” Indolizine Biosynthesis Reprinted from: Catalysts 2020 , 10 , 629, doi:10.3390/catal10060629 . . . . . . . . . . . . . . . . . . 203 Jos ́ e G. Sampedro, Miguel A. Rivera-Moran and Salvador Uribe-Carvajal Kramers’ Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity Reprinted from: Catalysts 2020 , 10 , 659, doi:10.3390/catal10060659 . . . . . . . . . . . . . . . . . . 219 Shang-Ming Huang, Hsin-Yi Huang, Yu-Min Chen, Chia-Hung Kuo and Chwen-Jen Shieh Continuous Production of 2-Phenylethyl Acetate in a Solvent-Free System Using a Packed-Bed Reactor with Novozym R © 435 Reprinted from: Catalysts 2020 , 10 , 714, doi:10.3390/catal10060714 . . . . . . . . . . . . . . . . . . 239 Adama A. Bojang and Ho Shing Wu Characterization of Electrode Performance in Enzymatic Biofuel Cells Using Cyclic Voltammetry and Electrochemical Impedance Spectroscopy Reprinted from: Catalysts 2020 , 10 , 782, doi:10.3390/catal10070782 . . . . . . . . . . . . . . . . . . 253 Thi Huong Ha Nguyen, Su-Min Woo, Ngoc Anh Nguyen, Gun-Su Cha, Soo-Jin Yeom, Hyung-Sik Kang and Chul-Ho Yun Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants Reprinted from: Catalysts 2020 , 10 , 823, doi:10.3390/catal10080823 . . . . . . . . . . . . . . . . . . 273 vi About the Editors Chia-Hung Kuo (Ph.D.) received his MS in Food Science and Technology from the National Taiwan University, Taiwan and a Ph.D. in Chemical Engineering from the National Taiwan University of Science and Technology, Taiwan. He is currently an associate professor of Seafood Science at the National Kaohsiung University of Science and Technology (NKUST), Taiwan. He has received several awards including the Outstanding New Teacher Award (2016), Outstanding Research Award (2018), and Special Outstanding Research Talent Award (2018–2019) from NKUST, the second largest university in Taiwan. He has also served as a Director of the Center for Aquatic Products Inspection Service at NKUST. He has more than 50 papers published in international journals and indexed on Scopus. His main research interests focus on process biochemistry, food engineering, extraction, oil and fat processing, and fermentation biotechnology. Chwen-Jen Shieh (Ph.D.) holds a Ph.D. in Food Science and Technology from the University of Georgia, USA. He is currently a distinguished professor of Biotechnology Center at the National Chung-Hsing University, Taiwan. He was also an outstanding research professor at Dayeh University, and a Director of R&D at Taisun Enterprise Company, Taiwan. His main research interests focus on biodiesel, lipid biocatalysis, enzyme technology, bioprocess optimization, supercritical fluid technology, and Chinese herb medicine biotechnology. vii catalysts Editorial Biocatalytic Process Optimization Chia-Hung Kuo 1, * and Chwen-Jen Shieh 2, * 1 Department of Seafood Science, National Kaohsiung University of Science and Technology, Kaohsiung 811, Taiwan 2 Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan * Correspondence: kuoch@nkust.edu.tw (C.-H.K.); cjshieh@nchu.edu.tw (C.-J.S.); Tel.: + 886-7-361-7141 (ext. 23646) (C.-H.K.); + 886-4-2284-0450 (5121) (C.-J.S.) Received: 10 October 2020; Accepted: 25 October 2020; Published: 12 November 2020 Biocatalysis refers to the use of microorganisms and enzymes in chemical reactions, has become increasingly popular and is frequently used in industrial applications due to the high e ffi ciency and selectivity of biocatalysts. Enzymes are e ff ective and precise biocatalysts as they are enantioselective, with mild reaction conditions, and are important tools in green chemistry. Biocatalysis is widely used in the pharmaceutical, food, cosmetic, and textile industries. Biocatalytic processes include enzyme production, biocatalytic process development, biotransformation, enzyme engineering, immobilization, and the recycling of biocatalysts. Factors a ff ecting biocatalytic reactions include substrate concentration, product concentration, enzyme or microorganism stability, inhibitors, temperature, and pH. As such, the optimization of biocatalytic processes is an important issue. Active compounds in natural products usually contain glycosides, which can be cleaved by glycoside hydrolases to increase biological activity. The cocktail enzyme cellulase has been used in the deglycosylation of piceid to produce resveratrol [ 1 ]. Rha et al. [ 2 ] tested several commercial food-grade enzymes for producing flavonol aglycones from green tea extracts via deglycosylation. Tannin acyl hydrolase and glycoside hydrolase activities in Plantase-CF (a multi-functional food-grade enzyme from Aspergillus niger with the ability of cellulolytic hydrolysis of carbohydrates) trigger the degalloylation of catechins and produce flavonol aglycones from green tea extracts. Optimal conditions for producing flavonol aglycones are pH 4.0 and 50 ◦ C. Biocatalysis can prevent catechin degradation, unlike hydrochloride treatment where 70% ( w / w ) of catechins disappear. Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic bonds in glycosides and have many applications, including the production of agaro-oligosaccharides, glucose, xylose, and xylobiose. Jiang et al. [ 3 ] reported a novel technique for producing agarotriose and agaropentaose from agaro-oligosaccharide using β -agarase. The results showed that 1.950 U / mL β -agarase AgWH50B was optimal for the preparation of agarotriose from the hydrolysis of agaroheptaose or agarononose, while 0.1900 U / mL β -agarase DagA was optimal for the preparation of agarotriose and agaropentaose from the hydrolysis of agaroheptaose or agarononose. In this study, the authors obtained value-added oligosaccharides from agarose by using di ff erent agarolytic enzymes or varying the enzyme amount. Aksenov et al. [ 4 ] used cellulolytic enzymes (mainly cellulases and xylanase) from recombinant Penicillium verruculosum to hydrolyze hardwood and softwood pulp, though the lignin content and drying process decreased the bioconversion of pulp to glucose. It was determined that fiber morphology, di ff ering xylan and mannan content, and hemicellulose localization in kraft fibers deeply a ff ected the enzymatic hydrolysis of bleached pulp. At a concentration of 10%, never-dried bleached kraft pulp demonstrated highly e ffi cient bioconversion, resulting in a concentration of more than 50 g / L sugar. Enzymatic biofuel cells rely on enzymes rather than conventional noble metal catalysts. Commonly used redox enzymes include glucose dehydrogenase, glucose oxidase (GOx), lacase (LAc), fructose dehydrogenase, and alcohol dehydrogenase [ 5 ]. Bojang and Wu [ 6 ] established the use of Catalysts 2020 , 10 , 1303; doi:10.3390 / catal10111303 www.mdpi.com / journal / catalysts 1 Catalysts 2020 , 10 , 1303 GOx / LAc modified electrodes as bioanodes and biocathodes for biofuel cells. Electrochemical analysis methods including cyclic voltammetry, the Nicholson method, the Randles–Sevcik equation, and electrochemical impedance spectroscopy were used to evaluate the performance of prepared electrodes. Following testing, the optimal bioanode and biocathode were determined to be a carbon paper–GOx–mediator–carbon nanotube with a current density of 800 μ A / cm 2 and a carbon paper–Lac–mediator–carbon nanotube with a current density of 600 μ A / cm 2 , respectively. The construction and use of enzyme electrodes can be applied to biofuel cells, bioreactors, biosensors, and micro-reactors. The disaccharide trehalose, a natural biostructure stabilizer that accumulates in the cytoplasm under stress conditions, is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants [ 7 ]. Sampedro et al. [ 8 ] studied the e ff ect of trehalose on enzyme reactions using Kramers’ theory. The role of trehalose was reviewed and the molecular interactions of trehalose–water–enzymes / proteins were described in detail, supported by recent in vitro and in silico experimental results. Importantly, the concept of coupling the enzyme’s structural dynamics to medium viscosity, as described by Kramers’ theory, is the central thesis of this paper and the focus is on enzyme catalysis. As such, the application of Kramers ́ theory is reinforced by relating the rate of inactivation, unfolding, and folding of enzymes to trehalose viscosity. The recently observed e ff ects of trehalose viscosity on DNA and RNA folding is mentioned as a corollary. Proteases belong to the hydrolase class of enzymes and hydrolyze proteins into smaller polypeptides or single amino acids. Protein hydrolysates have many biological functions and demonstrate antioxidant activity [ 9 , 10 ]. Zhang et al. [ 11 ] used pepsin, trypsin, dispase, papain, and bromelin to digest Pleurotus ostreatus protein extract (POPE). The antioxidant activity of the protein hydrolysates resulting from five di ff erent proteases were compared. The results showed that POPEP (POPE hydrolyzed by pepsin), with a molecular weight of 3–5 kDa, had the strongest antioxidant activity. Excessive free radicals or reactive oxygen species (ROS) are harmful to the human body since these components may destroy the normal functions of cells, tissues, and organs [ 12 ]. Superoxide dismutase, glutathione peroxide, and catalase can remove free radicals to reduce the risk of oxidative damage during periods of increased ROS. Mice pretreated with POPEP (3–5 kDa) showed significantly increased superoxide dismutase and glutathione peroxide enzyme activity in the liver, demonstrating that POPEP could protect the liver from oxidative damage. Sadauskiene et al. [ 13 ] reported on the e ff ects of long-term supplementation with aluminum (Al) or selenium (Se) on antioxidant enzyme activity in the brains and livers of mice. The results showed that 8 weeks of exposure to Se caused a statistically significant increase in superoxide dismutase, catalase, and glutathione reductase activities in the brain and / or liver, but the changes were dose-dependent. Exposure to Al caused a statistically significant increase in glutathione reductase activity in both organs. Protein hydrolysates containing bioactive peptides can be used to formulate nutraceuticals or functional ingredients in food. Cheng et al. [ 14 ] used three commercial proteases (alcalase, bromelain, and papain) to obtain eel protein hydrolysates (EPHs) from whole eels ( Anguilla marmorata ). The emulsion activity index (EAI) and emulsion stability index (ESI) of each EPH was determined to test the product stability. The EPH obtained from the treatment with alcalase showed optimal EAI and ESI and demonstrated antioxidant activity. The results indicated that alcalase-hydrolyzed EPH had good emulsifying properties and solubility, making it useful in food processing. The use of biocatalysis or biotransformation to produce pharmaceutical components has become a hot topic in biotechnology research. There are two main types of biocatalysts: whole cells and free enzymes. Both use enzymes to complete the reaction but in the former, the enzyme remains within the microorganism whereas for the latter, the enzyme has been separated and purified. When producing drugs and their intermediates, the most significant di ff erence between biocatalysis and traditional chemical methods is that the former is very e ff ective in the asymmetric synthesis of chiral compounds. In this Special Issue, several studies used biocatalysis to synthesize special compounds. Calycosin-7-O- β -D-glucoside is an isoflavonoid glucoside and one of the principal components of 2 Catalysts 2020 , 10 , 1303 Radix astragali , a well known medicinal and edible herb cited in European, Japanese, and Chinese literature. Hu et al. [ 15 ] used uridine diphosphate-dependent glucosyltransferase to glucosylate the C7 hydroxyl group of calycosin and synthesize calycosin-7-O- β -D-glucoside. Optimal conditions for batch production were determined, including the temperature, pH, and the concentrations of dimethyl sulfoxide, uridine diphosphate, sucrose, and calycosin. Eggerichs et al. [ 16 ] used styrene and indole monooxygenase to activate double bonds via chiral epoxidation. The reaction conditions were successfully optimized for two flavins containing two-component monooxygenases during the conversion of large hydrophobic styrene derivatives in the presence of organic cosolvents. Nguyen et al. [ 17 ] used Bacillus megaterium CYP102A1 monooxygenase for the regioselective hydroxylation of naringin dihydrochalcone to produce neoeriocitrindihydrochalcone. Kinetic parameters were used to compare the efficiency of dihydrochalcone hydroxylation by different CYP102A1 mutations. The indolizine core is present in many biologically active compounds and can be considered the sca ff olding in the preparation of new pharmaceuticals. Indolizines have been synthesized by lipases from Candida antarctica [ 18 ]. Botezatu et al. [ 19 ] used whole cells to catalyze a multicomponent reaction of activated alkynes, α -bromo-carbonyl reagents and 4,4 ′ -bipyridine in the synthesis of bis-indolizines. Several yeast strains were tested to evaluate the e ff ect of the reactants on their physiological activity. The optimal strain was Yarrowia lipolytica , which is an e ff ective biocatalyst in cycloaddition reactions and can be used to synthesize indolizines. In recent years, there has been a widespread use of immobilized lipase to catalyze specific reactions in the production of valuable molecules, such as nutraceutical and pharmaceutical compounds [ 20 , 21 ]. Chung et al. [ 22 ] used a surface-display system for the expression of lipase A in an E. coli expression system. It was reported that lipase A activity was low at lower shaking rates due to the limited amount of dissolved oxygen, while higher shaking rates increased shear stress, leading to a decrease in the specific activity. This phenomenon was confirmed using kinetic studies and it was established that cultivating lipase A at a moderate shaking speed optimized hydrolysis. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters are medicines used in the treatment of arteriosclerosis and hyperlipidemia. Kuo et al. [ 23 ] studied the lipase-catalyzed synthesis of DHA + EPA ethyl esters via the acidolysis of ethyl acetate with DHA + EPA concentrates. Lipase-catalyzed acidolysis has the advantage of not only synthesizing DHA + EPA ethyl ester e ffi ciently, but also allowing for the easy recovery of the product. Moreover, a response surface methodology (RSM) approach for the evaluation of the kinetic model was successful integrated with the rate equation to simulate the performance of the batch reactor. The integral equation showed a good predictive relationship between the simulated and experimental results. Conversion yields of 88%–94% were obtained for 100–400 mM DHA + EPA concentrate at a constant enzyme activity of 200 U, substrate ratio of 1:1 (DHA + EPA: EA), and reaction time of 300 min. Rychlicka et al. [ 24 ] developed a biotechnological method of synthesizing 3,4-dimethoxycinnamoylated phospholipids via the interesterification of egg-yolk phosphatidylcholine with the ethyl ester of 3,4-dimethoxycinnamic acid. RSM and a Box–Behnken design were used to evaluate reaction conditions. The optimal incorporation of 3,4-dimethoxycinnamic acid into phospholipids reached 21 mol%. Moreover, 3,4-dimethoxycinnamoylated lysophosphatidylcholine and 3,4-dimethoxycinnamoylated phosphatidylcholine were obtained in isolated yields of 27.5% and 3.5% ( w / w ), respectively. Huang et al. [ 25 ] developed a biocatalytic process for synthesizing rose-flavored ester-2-phenylethyl acetate using a packed-bed bioreactor system. The synthesis process was performed in a solvent free system, which is an environmentally friendly process. The optimization of the synthesis reaction was carried out by a three-level-three-factor Box–Behnken design and RSM. This continuous process can be applied to the environmentally friendly production of natural flavor compounds, such as rose aromatic esters. Grabner et al. [ 26 ] developed a continuous process for the synthesis of a statin side chain precursor using a deoxyribose-5-phosphate aldolase-catalyzed stereoselective aldol addition reaction. A series of substrates was tested but only acetaldehyde and chloroacetaldehyde gave reasonable results. An experimental design was used to optimize pH value, temperature, and flow 3 Catalysts 2020 , 10 , 1303 conditions. The immobilization alginate was chosen and the reaction rates of both alginate beads and the alginate–lu ff a matrix were tested. The optimized flow process (0.1 mL / min, 0.25 M of chloroacetaldehyde, and 0.5 M of acetaldehyde) produced 4.5 g of product per day in a bench-top reactor. Du et al. [ 27 ] developed a continuous-flow procedure for the synthesis of β -amino acid esters via the lipase-catalyzed Michael reactions of various aromatic amines with acrylates. Seventeen β -amino acid esters were rapidly synthesized by lipase TL IM from Thermomyces lanuginosus in continuous-flow microreactors. Optimal reaction parameters were determined, including the reaction medium, temperature, enzyme, substrate molar ratio, residence time / flow rate, and substrate structure. The salient features of this study are the green reaction conditions (using methanol as reaction medium), short residence time (30 min), and high yield. Several articles in this Special Issue have demonstrated that a bioreactor with immobilized enzymes is suitable for use in biocatalysis to synthesize active pharmaceutical ingredients, drug precursors, and value-added chemicals. The benefits of continuous flow biocatalysis, including improved reaction rates, in-line product removal and purification, better mixing, improved control, and improved enzyme stability, ultimately minimize labor and reduce production costs. The increased demand for greener and more cost-e ff ective processes will drive the rapid expansion of continuous flow biocatalysis in the next few years. In conclusion, this Special Issue shows that an optimized biocatalysis process can provide an environmentally friendly, clean, highly e ffi cient, low cost, and renewable process for the synthesis and production of valuable products. With further development and improvements, more biocatalysis processes may be applied in the future. Author Contributions: C.-H.K. and C.-J.S. prepared the article. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kuo, C.-H.; Chen, B.-Y.; Liu, Y.-C.; Chen, J.-H.; Shieh, C.-J. Production of resveratrol by piceid deglycosylation using cellulase. Catalysts 2016 , 6 , 32. [CrossRef] 2. Rha, C.-S.; Kim, S.-W.; Byoun, K.H.; Hong, Y.D.; Kim, D.-O. Simultaneous optimal production of flavonol aglycones and degalloylated catechins from green tea using a multi-function food-grade enzyme. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 5 catalysts Article Simultaneous Optimal Production of Flavonol Aglycones and Degalloylated Catechins from Green Tea Using a Multi-Function Food-Grade Enzyme Chan-Su Rha 1, *, Shin-Woo Kim 2 , Kyoung Hee Byoun 3 , Yong Deog Hong 1 and Dae-Ok Kim 4,5 1 Basic Research and Innovation Institute, Amorepacific R&D Center, Yongin 17074, Korea; hydhong@amorepacific.com 2 Research and Development Division, Bision Corp., Seoul 05854, Korea; swkim@bision.co.kr 3 Safety and Regulatory Research Institute, Amorepacific Corporation R&D Center, Yongin 17074, Korea; silling@amorepacific.com 4 Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Korea; DOKIM05@khu.ac.kr 5 Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Korea * Correspondence: teaman@amorepacific.com; Tel.: + 82-31-280-5981 Received: 14 September 2019; Accepted: 13 October 2019; Published: 16 October 2019 Abstract: (1) Background: Green tea (GT) contains well-known phytochemical compounds; namely, it is rich in flavan-3-ols (catechins) and flavonols comprising all glycoside forms. These compounds in GT might show better biological activities after a feasible enzymatic process, and the process on an industrial scale should consider enzyme specificity and cost-e ff ectiveness. (2) Methods: In this study, we evaluated the most e ff ective method for the enzymatic conversion of flavonoids from GT extract. One enzyme derived from Aspergillus niger (molecular weight 80–90 kDa) was ultimately selected, showing two distinct but simultaneous activities: intense glycoside hydrolase activity via deglycosylation and weak tannin acyl hydrolase activity via degalloylation. (3) Results: The optimum conditions for producing flavonol aglycones were pH 4.0 and 50 ◦ C. Myricetin glycosides were cleaved 3.7–7.0 times faster than kaempferol glycosides. Flavonol aglycones were produced e ff ectively by both enzymatic and hydrochloride treatment in a time-course reaction. Enzymatic treatment retained 80% ( w / w ) catechins, whereas 70% ( w / w ) of catechins disappeared by hydrochloride treatment. (4) Conclusions: This enzymatic process o ff ers an e ff ective method of conditionally producing flavonol aglycones and de-galloylated catechins from conversion of food-grade enzyme. Keywords: catechin; degalloylation; flavonol; glycoside hydrolase; optimization; tannase 1. Introduction Green tea (GT) is well-known to be enriched in catechins with flavonols / flavones as the second-most dominant flavonoids; these include myricetin, quercetin, apigenin, and kaempferol [ 1 ]. GT generally contains approximately 15% catechins and 0.4% flavonols on a dry weight basis [ 2 ]. Many studies have demonstrated that catechins are major sources of the vast diversity of GT bioactivities [ 3 , 4 ]. However, in human nutrition, GT flavonols are generally considered to be less crucial for the utility and functionality of GT. The content and compositions of flavonol and flavone glycosides vary according to GT cultivar [ 5 , 6 ]. Similarly, glycosylated flavonols and flavones have di ff erent sugar bonds and compositions according to the plants [ 7 ]. Flavonol glycosides consist of various sugar units with – O - or – C -conjugation on flavonol molecules. The glycosidic structure of flavonols a ff ects their biological and physiological properties, such as digestive stability and bioaccessibility [ 8 – 10 ]. For example , quercetin glycosides could be hydrolyzed to aglycone in the intestine [ 8 ], and quercetin 3-glucoside and quercetin 4-glucoside can be completely digested in humans [ 9 ]. However, flavonol Catalysts 2019 , 9 , 861; doi:10.3390 / catal9100861 www.mdpi.com / journal / catalysts 7 Catalysts 2019 , 9 , 861 glycosides with more complex structures undergo less hydrolysis in digestive conditions [ 10 ]. To date , the majority of research on the functionalities of flavonols has focused on the aglycone forms. Xiao et al. [11] proposed that flavonol aglycones have a higher a ffi nity for proteins due to their hydrophobic characteristics, allowing for easy absorption by cells. Moreover, flavone aglycones exhibited more potent anti-inflammatory e ff ects than their corresponding glycosides [ 12 ]. Flavonols, including myricetin, quercetin, and kaempferol , are known to possess numerous beneficial activities, such as antioxidative, anticancer, and antihyperlipidemic e ff ects [ 13 – 15 ]. Furthermore, flavonol supplementation was proven to potentially reduce the risk of cardiometabolic disease in a clinical trial [16]. Plumb et al. [ 17 ] presented that the antioxidant activities of GT flavonol glycosides were lower than those of their corresponding aglycones. For example, quercetin rhamnoside, a GT flavonol glycoside, was found to have an a ffi nity to bovine serum albumin 5600-fold lower than its corresponding aglycone, quercetin [ 11 ]. Owing to such limitations in the utilization of flavonols in human nutrition, a feasible enzymatic process was developed to break down plant-based flavonoid glycosides to their aglycones [ 18 ]. For example, the antioxidant capacity of soybean flour was enhanced by the enzymatic hydrolysis of phenolic glucoside in solid-state fungi fermentation [ 19 ]. In particular, tannase ( EC 3.1.1.20 ; tannin acyl hydrolase) shows good ability in the bioconversion of green tea extract (GTE), resulting in gallic acid (GA) and degalloylated catechins [20,21]. Increasing the content of GA through the conversion of GTE by tannase has been shown to improve the radical scavenging activities of GTE [ 22 ]. Moreover, conversion of (–)-epigallocatechin gallate (EGCG) by tannase attenuated its toxicity without a ff ecting the antiproliferative e ff ects [ 21 ]. Another study demonstrated that tannase-treated catechins influenced the expression of genes involved in the sodium-glucose transport proteins [ 23 ]. For their e ff ective absorption and utilization in the human body, some flavonol glycosides must be converted to their aglycone forms by digestive enzymes. Only 2% of the dietary flavonols that are digested and absorbed in the duodenum ultimately reach the plasma in intact form [ 24 ]. Flavonol glycosides are hydrolyzed by mammalian glucosidase in the small intestine before being absorbed in aglycone forms [ 25 ]. Some of the flavonol glycosides that are not hydrolyzed in the small intestine then move to the large intestine, where they undergo further metabolic reactions through bioconversions mediated by intestinal microorganisms [ 26 ]. Approximately 65% of human adults are estimated to have downregulated production of intestinal lactase (lactase phlorizin hydrolase; LPH) [ 27 ]. Lactase catalyzes the hydrolysis of β -glucosides, including phlorizin and flavonoid glucosides [ 28 ]. Furthermore, it was previously reported that flavonol-enriched fractions of GTE enhanced the bioavailability of the catechin epimers of GTE by downregulating the expression of the catechol- O -methyltransferase gene [ 29 ]. To date, research on the bioconversion of flavonol glycosides in GT to verify whether flavonol glycosides or aglycones are more beneficial to human nutrition and health is scarce. Other than tannase-based research, there are limited studies on the bioconversion of GT flavonoids by food-grade enzymes that show high specificity to flavonol glycosides. Since the enzymes for hydrolyzing glycosides are very diverse and specific to the type of glycosidic bond, multiple enzymes generally need to be used to accomplish complex food treatments. We hypothesized that a few multi-activity enzymes could be used to improve the e ffi ciency of conversion for obtaining functional compounds with nutritional benefits. To test this possibility, we screened multi-functional food-grade enzymes among eight kinds of enzymes from a broth of fungi. We then selected an enzyme and further evaluated its kinetic characteristics and compared the e ff ectiveness of enzymatic treatment for producing flavonol aglycones from flavonol glycosides in GTE to provide an optimized method. 8 Catalysts 2019 , 9 , 861 2. Results 2.1. Composition Changes of GTE by Various Food–Grade Enzymes The tested commercial food-grade enzymes exhibited various multi-functional activities (Table 1). Although all of the enzyme